Project summary: The objective of the proposed study is to develop broadly effective attenuation strategies for the rational design of live-attenuated vaccines (LAVs) against flaviviruses. Pathogenic flaviviruses cause severe human diseases such as hemorrhagic fever and encephalitis. The use of two legacy vaccines, yellow fever virus (YFV) 17D and Japanese encephalitis virus SA14-14-2, has demonstrated how immunization can be an efficient strategy for disease control. However, the empirical approach used for the development of these LAVs has proven ineffective in producing candidate LAVs for other flaviviruses, thereby demanding new strategies for rational vaccine design. Previously, the rational design of flavivirus LAVs was based on the introduction of mutations that lead to attenuated phenotypes observed in the two legacy vaccines and other attenuated mutants. This failed to produce broadly effective attenuation concepts, because the mutagenesis targets lacked conserved sequences or interacted with diverse host molecules. A major challenge in designing LAVs is the field's limited knowledge of flavivirus virulence mechanisms. Target genes that contain consensus sequences, functionally important for the virulence of different flaviviruses, are yet to be identified. In this study, the PI will develop two attenuation strategies by interfering with the interdomain movements of flavivirus envelope (E) proteins. Interdomain movements of E proteins are universally conserved mechanisms in all flaviviruses and critical for viral membrane fusion and virion assembly; they are controlled by highly conserved sequences in two sets of interdomain peptides, the envelope protein domain I (EDI) ? envelope protein domain II (EDII) hinge and the EDI ? envelope protein domain III (EDIII) linker. The central hypothesis is that conserved residues in the interdomain peptides are functionally important for flavivirus virulence, regardless of their tissue tropism and disease pathogenesis. To remove virulence determinants in the two interdomain peptides, mutagenesis analyses will be conducted in the following two specific aims: In Aim 1, highly conserved hydrophobic residues will be mutated to interfere with the hydrophobic interactions that contribute to EDI-EDII hinge structure and functions; in Aim 2, the structures and functions of EDI-EDIII linker will be disrupted by removing functionally important side-chains of conserved residues and inserting additional glycine/prolin residues to increase peptide flexibility. Broadly effective attenuation strategies will be developed by engineering selected mutations into the full-length complementary DNA infectious clones of two model flaviviruses, West Nile virus and YFV, and then demonstrating the loss of virulence in respective mouse models. The completion of the proposed study will lead to an advancement in knowledge regarding the functional importance of the EDI-EDII hinge and EDI-EDIII linker interdomain regions for flavivirus virulence. The results of this study are expected to provide the basis for broadly effective attenuation strategies for pathogenic flaviviruses and facilitate the rational design of future candidate flavivirus LAVs.